The major goal of mitosis is to distribute the genetic material accurately between the two daughter cells. Defects in meiosis or mitosis lead to aneuploidy, which is a significant cause of birth defects and is a hallmark of tumorigenesis. Proper spindle function relies on precise spatial and temporal control of microtubule (MT) dynamics and the integration of forces of motor proteins. Defects in regulated MT dynamics lead to spindle multi-polarity, improper kinetochore-MT attachments, delayed mitotic progression, and improper chromosome segregation. Despite the generation of an extensive parts list for the spindle, a major unanswered question is to understand how MT dynamics and motor protein activity are spatially and temporally regulated to ensure proper spindle architecture and function. This has been due, in part, to a lack of appropriate tools that could be used to relate key regulatory biochemical events to where those events are controlled spatially in the spindle. Our recent implementation of new FRET-based biosensors combined with FLIM and super resolution microscopy is now enabling us to address this important question. In this proposal we will: 1) Define how MT sliding is spatially modulated to control spindle organization by reconstituting MT sliding, and using FLIM imaging of novel intermolecular FRET reporters. These experiments will establish a paradigm for how the spatial control of motor activity contributes to the global organization of the spindle and will also define the critical features of XCTK2 that contribute to pole focusing and could be targeted for therapeutic development for treatment of tumors with centrosome amplification. 2) Define how Aurora B kinase modulates protein conformation, targeting, and function of the mitotic kinesin MCAK by testing how the CT of MCAK modulates conformation and activity. We will use Xenopus spindle assembly assays with various MCAK CT mutants and Fluorescence Lifetime Imaging of our MCAK biosensors to define where and when this conformational regulation occurs. This work will define spatial control mechanisms for the control of MCAK that will also serve as a model for regulation of protein activity by kinase networks. 3) Define how the integration of spatially distinct MT populations contributes to mitotic fidelity by testing the model that excess polymerization of non-kinetochore MTs increases mal-attached kinetochores and reduces mitotic fidelity. 3D-SIM super-resolution imaging of chromosome- MT interactions after kinesin knockdown will allow us to visualize how loss of these proteins lead to mal- oriented chromosomes. In vitro reconstitution assays using single molecule imaging and PALM/STORM will define how different MT dynamics regulators cooperatively control plus-end dynamics. Together these studies will bridge the gap in knowledge between how biochemical activities in vitro impact spindle structure and function in vivo.